Third-generation sequencing approaches are largely focusing on single-molecule strategies with the ability to achieve long read lengths. Single-molecule approaches require little or no sample preparation, saving time and reagent costs. They are more accurate since there is less chance of errors as no amplification is needed and there is no bias in molecular quantification. In addition, single-molecule techniques allow direct sequencing of mRNA, allowing understanding of post-transcription editing variations and copy-number studies. Ideally, single-molecule SBS can be massively-parallel and real-time, operating at synthesis rates as high as 1 msec for DNA polymerase, however complex optics required to collect photons efficiently make scaling of the platforms to high densities difficult. A promising route for overcoming the challenges to optical techniques is bioelectronic detection. The direct, real-time detection of this reaction product by electrical means represents a two-fold challenge. First, the minute amount of charge involved falls well below the noise floor for solid-state detection. Second, the presence of a high concentration of screening ions in physiological buffers greatly reduces the range and strength of electrostatic interactions. As a result, conventional electrical detection strategies, including impedance spectroscopy, field-effect detection and Faradaic reactions, lack sufficient sensitivity to detect single molecules. In this four-year effort, we develop a real-time, single-molecule sequencing approach based on the electrical detection of specifically engineered electrochemical tags that are attached to each of the four nucleotides. A base-specific electrochemical tag is released during the nucleotide incorporation; this tag is then activated through a phosphatase reaction to become redox active and is subsequently collected into a single molecule fingerprinting region (composed of four nanogap transducers). Redox cycling is used to produce an amplified signal for detection in the fingerprinting region. This approach to signal amplification is the electrical analog of fluorescen labels which see repeated excitation and emission under constant illumination to achieve detection gain. These nanogap transducers are integrated onto a CMOS integrated circuit in a highly multiplexed, parallel format. The proposed approach combines the advantages of single-molecule real time sequencing with a CMOS-compatible single molecule signal transduction platform and its attendant scalability benefits PUBLIC HEALTH RELEVANCE: Developing single-molecule, real-time electronic DNA sequencing systems will enable ultraportable and extremely cost effective full genome sequencing systems. Fast and low-cost full genome DNA sequencing has the potential to accelerate major advances in the understanding, diagnosis, treatment and prevention of disease, including cancer, and in the identification of pathogens in the environment, food and livestock.